A primary goal of genetic engineering is to obtain plants having improved characteristics or traits. Many different types of characteristics or traits are considered advantageous, but those of particular importance include resistance to plant diseases, resistance to viruses or insects and resistance to herbicides. Other advantageous characteristics or traits include tolerance to cold or soil salinity, enhanced stability or shelf life of the ultimate consumer product obtained from a plant, or improvement in the nutritional value of edible portions of a plant.
Recent advances in genetic engineering have enabled the incorporation of a selected gene (or genes) into plant cells to impart a desired quality (or qualities) to a plant of interest. The selected gene (or genes) may be derived from a source different from the plant of interest or may be native to the desired plant, but engineered to have different or improved qualities. This new gene (or genes) may then be expressed in cells of the regenerated plant to exhibit the new trait or characteristic.
In order for the newly incorporated gene to express the protein for which it codes in a plant cell, the proper regulatory signals must be present, in the proper location with respect to the gene. These regulatory signals include a promoter, a 5′ non-translated leader sequence and a 3′ polyadenylation signal.
The efficiency of gene expression is governed largely by the promoter used to express the gene. A promoter is typically a DNA sequence that directs the cellular machinery of a plant to produce (transcribe) RNA (transcript) from a contiguous transcribable region downstream (3′) of the promoter. The promoter influences the rate at which the transcript of the gene is made. Assuming the transcript includes a coding region with appropriate translational signals, the promoter also influences the rate at which the resultant protein product of the gene is produced. Promoter activity also can depend on the presence of several other cis-acting regulatory elements which, in conjunction with cellular factors, determine strength, specificity, and transcription initiation site (for a review, see Zawel and Reinberg, 1992, Curr. Opin. Cell Biol. 4: 488).
It has been shown that certain promoters are able to direct RNA synthesis at a higher rate relative to other promoters. These are called “strong promoters”. Certain other promoters have been shown to direct RNA production at higher levels only in particular types of cells or tissues and are often referred to as “tissue-specific promoters”. Promoters that are capable of directing RNA production in many or all tissues of a plant are called “constitutive promoters”. Thus, expression of a chimeric gene (or genes) introduced into a plant may potentially be controlled by identifying and using a promoter with the desired characteristics.
There have been numerous promoters described for gene expression in dicotyledonous plants. However, there still remains a dearth of promoters that can be used for effective expression of foreign or endogenous coding sequences in monocotyledonous plants.
One promoter that has been used widely for directing gene expression in, especially, dicotyledonous plants, is the 35S promoter derived from cauliflower mosaic virus (CaMV), a member of the family Caulimoviridae. CaMV is the source for both the 35S and 19S promoters, but the 35S promoter has been most widely used. It is capable of directing strong, constitutive expression in many dicotyledonous species and is generally functional, albeit with somewhat reduced activity, in monocotyledonous plants (Benfey and Chua, Science 244:174-181, 1989; Science 250: 959-966, 1990; Fang et. al., Plant Cell 1:141-150, 1989; Fütterer et al., EMBO J 9: 1697-1707, 1990; Terada and Shimamoto, Mol. Gen Genet 220:389-392, 1990).
In addition to the caulimoviruses, promoters have also been isolated from several badnaviruses including Commelina yellow mottle virus, banana streak virus and sugarcane bacilliform virus. Badnaviruses are pararetroviruses, also classified in the family Caulimoviridae, genus Badnavirus. They have bacilliform-shaped virions approximately 30×130 nm encapsidating a circular dsDNA genome ranging from 7.1-7.6 kbp that contains three open reading frames (ORF's) (Lockhart B. E. L. and Olszewski N. E. in Ganry J. (ed). CIRAD/INIBAP, Montpellier, 1993, pp. 105-113; Lockhart et al., (eds). Virus Taxonomy: Sixth Report of the international Committee on the Taxonomy of Viruses. Springer, Wien, 1995, pp. 185-188; Medberry et al., Nucleic Acids Research 18:5505-5513, 1990.). Badnaviruses have been reported to infect a wide range of tropical plant species, including economically important monocotyledonous crops such banana, sugarcane and rice.
Nevertheless, despite the existence of these representative promoters, there remains a need to identify promoter sequences with improved efficacy and efficiency in directing the expression of heterologous DNA sequences in all plant species and, in particular, in monocotyledonous plants.